Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein.

Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ((59)Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca(2+) uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca(2+) uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein.

Pathogenesis of lipid metabolism disorder in hepatitis C: polyunsaturated fatty acids counteract lipid alterations induced by the core protein.

BACKGROUND & AIMS: Disturbance in lipid metabolism is one of the features of chronic hepatitis C, being a crucial determinant of the progression of liver fibrosis. Experimental studies have revealed that the core protein of hepatitis C virus (HCV) induces steatosis. METHODS: The activities of fatty acid metabolizing enzymes were determined by analyzing the fatty acid compositions in HepG2 cells with or without core protein expression. RESULTS: There was a marked accumulation of triglycerides in core-expressing HepG2 cells. While the oleic/stearic acid (18:1/18:0) and palmitoleic/palmitic acid ratio (16:1/16:0) were comparable in both the core-expressing and the control cells, there was a marked accumulation of downstream product, 5,8,11-eicosatrienoic acid (20:3(n-9)) in the core-expressing HepG2 cells. The addition of eicosatetraynoic acid, which inhibits delta-6 desaturase activity which is inherently high in HepG2 cells, led to a marked accumulation of oleic and palmitoleic acids in the core-expressing cells, showing that delta-9 desaturase was activated by the core protein. Eicosapentaenoic acid (20:5(n-3)) or arachidonic acid (20:4(n-6)) administration significantly decreased delta-9 desaturase activity, the concentration of 20:3(n-9), and triglyceride accumulation. This lipid metabolism disorder was associated with NADH accumulation due to mitochondrial dysfunction, and was reversed by the addition of pyruvate through NADH utilization. CONCLUSIONS: The fatty acid enzyme, delta-9 desaturase, was activated by HCV core protein and polyunsaturated fatty acids counteracted this impact of the core protein on lipid metabolism. These results may open up new insights into the mechanism of lipid metabolism disorder associated with HCV infection and provide clues for the development of new therapeutic devices.

Hepatitis C virus core protein compromises iron-induced activation of antioxidants in mice and HepG2 cells.

One of the characteristics of hepatitis C virus (HCV) infection is the unusual augmentation of oxidative stress, which is exacerbated by iron accumulation in the liver, as observed frequently in hepatitis C patients. Using a transgenic mouse model, the core protein of HCV was shown previously to induce the overproduction of reactive oxygen species (ROS) in the liver. In the present study, the impact of iron overloading on the oxidant/antioxidant system was examined using this mouse model and cultured cells. Iron overloading caused the induction of ROS as well as antioxidants. However, the augmentation of some antioxidants, including heme oxygenase-1 and NADH dehydrogenase, quinone 1, was compromised by the presence of the core protein. The attenuation of iron-induced augmentation of heme oxygenase-1 was also confirmed in HepG2 cells expressing the core protein. This attenuation was not dependent on the Nrf2 transcription factor. Thus, HCV infection not only induces oxidative stress but also hampers the iron-induced antioxidant activation in the liver, thereby exacerbating oxidative stress that would facilitate hepatocarcinogenesis.

Tacrolimus ameliorates metabolic disturbance and oxidative stress caused by hepatitis C virus core protein: analysis using mouse model and cultured cells.

Hepatic steatosis and insulin resistance are factors that aggravate the progression of liver disease caused by hepatitis C virus (HCV) infection. In the pathogenesis of liver disease and metabolic disorders in HCV infection, oxidative stress due to mitochondrial respiratory chain dysfunction plays a pivotal role. Tacrolimus (FK506) is supposed to protect mitochondrial respiratory function. We studied whether tacrolimus affects the development of HCV-associated liver disease using HCV core gene transgenic mice, which develop hepatic steatosis, insulin resistance, and hepatocellular carcinoma. Administration of tacrolimus to HCV core gene transgenic mice three times per week for 3 months led to a significant reduction in the amounts of lipid in the liver as well as in serum insulin. Tacrolimus treatment also ameliorated oxidative stress and DNA damage in the liver of the core gene transgenic mice. Tacrolimus administration reproduced these effects in a dose-dependent manner in HepG2 cells expressing the core protein. The intrahepatic level of tumor necrosis factor-alpha, which may be a key molecule for the pathogenesis in HCV infection, was significantly decreased in tacrolimus-treated core gene transgenic mice. Tacrolimus thus reversed the effect of the core protein in the pathogenesis of HCV-associated liver disease. These results may provide new therapeutic tools for chronic hepatitis C, in which oxidative stress and abnormalities in lipid and glucose metabolism contribute to liver pathogenesis.

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein.

UNLABELLED: The hepatitis C virus (HCV) core protein is involved in viral pathogenesis such as oxidative stress induction and lipid metabolism disturbance, and is primarily located in the cytoplasm and endoplasmic reticulum in association with lipid droplets as well as in the mitochondria. To clarify the impact of the core protein on mitochondria, we analyzed the expression pattern of mitochondrial proteins in core protein-expressing cells by two-dimensional polyacrylamide gel electrophoresis. Several proteins related to the mitochondrial respiratory chain or protein chaperons were identified by mass spectrometry. Among the identified proteins with consistently different expressions, prohibitin, a mitochondrial protein chaperon, was up-regulated not only in core-expressing cells but also in full-genomic replicon cells and livers of core-gene transgenic mice. The stability of prohibitin was increased through interaction with the core protein. Further analysis demonstrated that interaction of prohibitin with mitochondrial DNA-encoded subunits of cytochrome c oxidase (COX) was disturbed by the core protein, resulting in a significant decrease in COX activity. CONCLUSION: The HCV core protein affects the steady-state levels of a subset of mitochondrial proteins including prohibitin, which may lead to an impaired function of the mitochondrial respiratory chain with the overproduction of oxidative stress.

Molecular basis for the synergy between alcohol and hepatitis C virus in hepatocarcinogenesis.

Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). In addition, heavy alcohol use has been linked with earlier progression to HCC in chronic hepatitis C patients. However, in the pathogenesis of HCV-associated HCC, it still remains controversial as to whether the virus plays a direct or an indirect role, and as to how alcohol operates in the acceleration of HCC development. Several studies using transgenic mouse models, in which the core protein of HCV has an oncogenic potential, indicate that HCV is directly involved in hepatocarcinogenesis, although other factors such as continuous inflammation or environmental factors seem also to play a role. The downstream events of the HCV core protein expression in the transgenic mouse HCC model are segregated into two pathways. One is the augmented production of oxidative stress in the absence of inflammation along with the attenuation of some scavenging systems in the putative preneoplastic stage with steatosis in the liver. The other pathway is the alteration in cellular gene expression and intracellular signaling, including the mitogen-activated protein kinase cascade. The combination of these pathways would explain the unusually high incidence and multicentric nature of HCC development in HCV infection. In addition, alcohol feeding in this animal model further activated the two pathways synergistically with HCV, leading to an earlier development of HCC. Such a synergy would reveal the molecular basis for the acceleration of HCC development by alcohol in HCV infection.

Prevalence of hepatitis B virus infection in Japanese patients with HIV.

Patients with HIV infection are frequently infected with hepatitis viruses, which are presently the major cause of mortality in HIV-infected patients after the widespread use of highly active antiretrovirus therapy. We previously reported that approximately 20% of HIV-positive Japanese patients were also infected with hepatitis C virus (HCV). Hepatitis B virus (HBV) infection may also be an impediment to a good course of treatment for HIV-infected patients, because of recurrent liver injuries and a common effectiveness of some anti-HIV drugs on HBV replication. However, the status of co-infection with HIV and HBV in Japan is unclear. We conducted a nationwide survey to determine the prevalence of HIV-HBV co-infection by distributing a questionnaire to the hospitals belonging to the HIV/AIDS Network of Japan. Among the 5998patients reported to be HIV positive, 377 (6.4%) were positive for the hepatitis B surface antigen. Homosexual men accounted for two-thirds (70.8%) of the HIV-HBV co-infected patients, distinct from HIV-HCV co-infection in Japan in which most of the HIV-HCV co-infected patients were recipients of blood products. One-third of HIV-HBV co-infected patients had elevated serum alanine aminotransferase levels at least once during the 1-year observation period. In conclusion, some HIV-infected Japanese patients also have HBV infection and liver disease. A detailed analysis of the progression and activity of liver disease in co-infected patients is needed.

Amino acid substitutions in the S region of hepatitis B virus in sera from patients with acute hepatitis.

BACKGROUND: An increase in the number of acute hepatitis patients with hepatitis B virus (HBV) of non-indigenous genotypes may reduce the efficacy of vaccination against HBV. METHODS: We have determined the amino acid (aa) sequences in the major hydrophilic region (MHR) in the S region of HBV in patients with acute hepatitis B and compared those with the ones from HBV strains used for the production of HBV vaccines commercially available in Japan. RESULTS: Of 48 patients studied, 11 were infected with genotype A, 11 with genotype B and 26 with genotype C HBV. The aa sequences of the nine genotype A isolates were the same as the aa sequence of J02205 which is used for the production of one of the commercially available recombinant vaccines. The aa sequences of the 11 genotype B isolates differed from the aa sequence of J02205 in two or three amino acids. Of the26 genotype C isolates, 22 had the same aa sequence as X01587 which is used for the production of another recombinant vaccine. The remaining genotype C isolates had aa substitutions at aa131, which have a potential to alter the hydropathy and the three-dimensional structure of the MHR. The differences among the three current HBV vaccines in aa sequences in the MHR theoretically alter the hydropathy and three-dimensional structure. CONCLUSION: Our results suggest that the transmission of HBV isolates with different genotypes or with aa substitutions in the MHR might reduce the efficacy of currently available HBV vaccines in the protection of HBV infections.

Oxidative stress and hepatitis C viral infection.

The involvement of oxidative stress in the pathogenesis of hepatitis and hepatocellular carcinoma has been strongly suggested. Oxidative stress is produced by inflammatory processes that occur in hepatitis via immunological mechanisms. In addition, in hepatitis C virus (HCV) infectious disease, some role has been assigned to viral proteins in the induction of oxidative stress. In the presence of hepatic steatosis, insulin resistance and increased levels of some cytokines, all of which are also induced by viral protein expression, oxidative stress is enhanced in HCV infection. In this sense, the role of oxidative stress in the progression of chronic hepatitis and hepatocarcinogenesis is greater in hepatitis C than in other types of hepatitis such as hepatitis B or autoimmune hepatitis. The additive effects of oxidative stress caused by the inflammatory process and that induced by HCV proteins may, furthermore, exert synergistic effects with alterations in intracellular signaling systems such as mitogen-activated protein kinases (MAPK), which are also induced by HCV proteins. These synergistic effects may be responsible for rare characteristics, that is, the high incidence and multicentric nature of hepatocarcinogenesis in HCV infection.

Hepatitis C virus core protein exerts an inhibitory effect on suppressor of cytokine signaling (SOCS)-1 gene expression.

BACKGROUND/AIMS: Suppressor of cytokine signaling (SOCS)-1, a negative feedback regulator of cytokine signaling pathway, also has a tumor suppressor activity, the silencing of its gene by hypermethylation is suggested to contribute to hepatocarcinogenesis. We studied the effect of the core protein of hepatitis C virus (HCV) on the expression of SOCS-1 gene. METHODS: HCV core gene transgenic mice, which develop hepatocellular carcinoma late in life, HepG2 cells expressing the core protein, and human liver tissues were analyzed. RESULTS: The expression of SOCS-1 gene was significantly suppressed in the liver of core gene transgenic mice and HepG2 cells expressing the core protein, while that of SOCS-3 gene was conserved. SOCS-1 expression levels also decreased in HCV-positive human liver tissues. The core protein differentially down-regulated the expression of signal transducer and activator of transcription (STAT) target genes, but rather enhanced STAT1 and STAT3 activation after interleukin-6 stimulation in mouse liver tissues and cells. CONCLUSIONS: HCV core protein down-regulates the expression of SOCS-1 gene. This is a mechanism leading to SOCS-1 silencing, an alternative to the hypermethylation of the gene; this effect of the core protein may modulate the intracellular signaling pathway, contributing to the pathogenesis in HCV infection including hepatocarcinogenesis.

Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma.

BACKGROUND: Silencing of the suppressor of cytokine signaling ( SOCS-1) by aberrant methylation at the CpG island in the coding region gene has been reported in hepatocellular carcinoma (HCC). However, principally, it is methylation in the 5'-noncoding region but not that in the coding region which determines the regulation of gene expression. METHODS: Methylation-specific PCR was performed for the analysis of methylation status both in the 5'-noncoding region and the CpG island of SOCS-1 from 22 HCC tissue samples with adjacent non-HCC tissue samples and from two cell lines. RESULTS: Using primers in the CpG island, 9 of 22 HCC samples exhibited aberrant methylation of SOCS-1, while only 1 of 22 adjacent non-HCC samples did so. The unmethylation pattern was detected in 1 of 22 HCC and in 5 of 22 non-HCC samples. Thus, aberrant methylation of SOCS-1 was significantly associated with HCC ( P = 0.0076 by Fisher's exact test). Using primers in the 5'-noncoding region, aberrant methylation was observed in 12 of 22 HCC and in 2 non-HCC samples. The unmethylated pattern was observed in 5 of 22 HCC and in 10 of 22 non-HCC samples ( P = 0.0042). There was no significant correlation between the methylation status of SOCS-1 and clinicopathological findings, such as the presence or absence of cirrhosis or the histological grade of HCC. CONCLUSIONS: Aberrant methylation of the SOCS-1 had a significant correlation with HCC. The rate of aberrant methylation was similar in the 5'-noncoding region and in the CpG island. Aberrant methylation of SOCS-1 may be associated with hepatocarcinogenesis, although further studies are necessary.

Hepatitis C virus infection and diabetes: direct involvement of the virus in the development of insulin resistance.

BACKGROUND & AIMS: Epidemiological studies have suggested a linkage between type 2 diabetes and chronic hepatitis C virus (HCV) infection. However, the presence of additional factors such as obesity, aging, or cirrhosis prevents the establishment of a definite relationship between these 2 conditions. METHODS: A mouse model transgenic for the HCV core gene was used. RESULTS: In the glucose tolerance test, plasma glucose levels were higher at all time points including in the fasting state in the core gene transgenic mice than in control mice, although the difference was not statistically significant. In contrast, the transgenic mice exhibited a marked insulin resistance as revealed by the insulin tolerance test, as well as significantly higher basal serum insulin levels. Feeding with a high-fat diet led to the development of overt diabetes in the transgenic mice but not in control mice. A high level of tumor necrosis factor-alpha, which has been also observed in human chronic hepatitis C patients, was considered to be one of the bases of insulin resistance in the transgenic mice, which acts by disturbing tyrosine phosphorylation of insulin receptor substrate-1. Moreover, administration of an anti-tumor necrosis factor-alpha antibody restored insulin sensitivity. CONCLUSIONS: The ability of insulin to lower the plasma glucose level in the HCV transgenic mice was impaired, as observed in chronic hepatitis C patients. These results provide a direct experimental evidence for the contribution of HCV in the development of insulin resistance in human HCV infection, which finally leads to the development of type 2 diabetes.

[Management of patients with hepatic metastases from gastric carcinoma].

Many studies have reported the benefit of hepatic resection for metastatic tumors from colorectal cancer. However, the significance of hepatic resection for gastric metastasis has been controversial. Peritoneal metastases were recognized in 40% of gastric cancer patients with liver metastases, and metastatic lesions in both lobes of the liver were seen in 60% of patients. Resection with curability B was performed in only 10% of the all gastric cancer patients with liver metastases. However, the overall 5-year survival rate of curability B resection was more than 30%, suggesting that it is worth while treating metastases of gastric cancer to the liver. Both synchronous and metachronous metastases are indications for hepatectomy. If there is only one liver metastasis, with no peritoneal and paraaortic lymph node metastases, curability B resection can be performed. Although there is no consensus on the method of hepatectomy, wedge resection is satisfactory. As systemic chemotherapy, S-1 + cisplatin results in a response rate of 50% in patients with metastases to the liver. As arterial infusion chemotherapy, the 5-fluorouracil-doxorubicin-mitomycin (FAM) regimen yields a response rate of more than 70% including 15% complete response rate. FAM is thus a superior regimen, but care must be taken to prevent complications resulting from intraarterial infusion of outside the vas due to deviation of the catheter.

Hepatitis C virus core protein activates ERK and p38 MAPK in cooperation with ethanol in transgenic mice.

In human chronic hepatitis C, alcohol intake is a synergistic factor for the acceleration of hepatocarcinogenesis. Recently, we showed a significant increase of reactive oxygen species (ROS) in hepatitis C virus (HCV) core-transgenic mice fed ethanol-containing diets. Because previous studies indicated that ROS is closely associated with mitogen-activated protein kinases (MAPK), we examined activities of c-Jun N-terminal kinase, p38 MAPK, and extracellular signal-regulated kinase (ERK) in the liver of core-transgenic and nontransgenic mice with short-term ethanol feeding. Activity of ERK and p38 MAPK was increased in core-transgenic mice compared with nontransgenic mice, whereas neither ERK nor p38 MAPK was activated in core-transgenic mice with normal diets. In addition, activity of cyclic-AMP and serum responsive element, downstream pathways of p38 MAPK and ERK, was also increased. Comparison of gene expression profiles by cDNA microarray and real-time PCR revealed that galectin-1, which is associated with cell transformation, was significantly increased in ethanol-fed core-transgenic mice. On the other hand, glutathione S-transferase (GST), which plays a key role in protecting cells from oxidative stress, was decreased. In conclusion, these results suggest that HCV core protein cooperates with ethanol for the activation of some MAPK pathways, and leads to the modulation of several genes, contributing to the pathogenesis of liver disease of HCV-infected patients with high ethanol consumption.

Suppression of oxidative stress-induced hepatocyte injury by calpain antisense.

BACKGROUND: Calpain, a cytosolic Ca(2+)-dependent proteinase, plays a pivotal role in cell injury. In this study, we investigated the effect of calpain-mu antisense oligonucleotide on oxidative stress induced-hepatocyte injury. MATERIALS AND METHODS: Hemagglutinating virus of Japan liposome complex with one of three types of antisense oligonucleotide (AS-1, AS-2, AS-3) or scramble oligonucleotide was added to the culture medium of HuH7 cells and incubated for 6 days. The expression of calpain-mu protein was examined by Western blotting. After the addition of tert-butyl hydroperoxide, bleb formation was examined by phase contrast microscopy, and cell viability was assessed by the release of lactate dehydrogenase. RESULTS: Incubation of HuH7 cells with AS-2 resulted in a decrease in the amount of calpain on day 4 and a further decrease to almost undetectable levels on day 6, whereas scramble oligonucleotide had no effect. Bleb formation was observed 120 min after the addition of tert-butyl hydroperoxide in scramble oligonucleotide-treated cells as in untreated cells. In contrast, it was rarely observed in AS-2-treated cells. Lactate dehydrogenase release was significantly suppressed in AS-2-treated cells, compared with that in scramble oligonucleotide treated-cells. CONCLUSIONS: Our findings suggest that calpain activation is involved in the pathogenesis of oxidative stress injury and that transfection of calpain antisense may potentially protect against ischemia/reperfusion liver injury.

Serum lipid profile of patients with genotype 1b hepatitis C viral infection in Japan.

Hepatitis C virus (HCV) infection is associated with the development of steatosis in the liver. Recently, infection with genotype 3a HCV has been reported to have a closer association with hepatic steatosis than that with genotype 1 or 2 HCV. Moreover, infection with genotype 3a HCV but not with genotype 1 has been shown to be associated with serum hypocholesterolemia or hypobetalipoproteinemia in European countries. We conducted a case control study to characterize the serum lipid profile in patients infected with genotype 1b HCV, which is the most prevalent HCV genotype in Japan. These patients had significantly lower serum cholesterol levels than those infected with HBV or genotype 2a HCV who had similar liver disease progression and body mass index. Further analysis of serum apolipoproteins revealed that not only apolipoprotein B but also apolipoprotein CII and apolipoprotein CIII levels were significantly reduced, while apolipoprotein AI, AII and E levels were similar in patients infected with genotype 1b HCV and those with HBV or genotype 2a HCV. These results indicate that, in Japan, infection with genotype 1b HCV is a cause of lipid metabolism disturbances, which may be associated with the pathogenesis of hepatitis C liver disease.